1. Technical Field
The present disclosure relates to a novel merocyanine-based compound capable of labeling biomolecules by intercalating biomolecules, and to a dye, kit and contrast medium composition for labelling biomolecules comprising the same.
2. Description of the Related Art
Fluorescent dyes have been used for the detection of a variety of biological samples involving nucleic acids, and nucleic acids including DNA and RNA. Accordingly, studies have been actively made on fluorescent nucleic acid dyes that are specifically bound or intercalated into nucleic acids to form a complex exhibiting excellent fluorescence.
Dyes specifically bound or intercalated into nucleic acids can be used in pure solutions, cell extracts, electrophoresis gels, microarray chips, living or immobilized cells, apoptotic cells and environmental samples, and can be used to detect the presence and amount of DNA and RNA in various samples. In particular, such fluorescent dyes can be used for the detection of nucleic acids through polymerase chain reaction (PCR), which is a typical method used in genome research and medical diagnosis.
In this case, since a quantitative PCR which is proportional to the amount of the sample nucleic acids is impossible in the case of general end-point PCR, real-time quantitative PCR (real-time PCR or qPCR) is mainly used for quantitative analysis of the amount of nucleic acids by measuring the fluorescence value changing in real time for each amplification cycle.
Such qPCR allows quantitative analysis by measuring the fluorescence signal from the PCR result. As a method for measuring the fluorescence signal, a detection method using a fluorescent probe and a detection method using an intercalating fluorescent dye are mainly known.
A detection method using a fluorescent probe uses a probe (for example, an oligonucleotide) labeled with a complex of a fluorescent dye and a quencher dye. When an oligonucleotide labeled with a complex of a fluorescent dye and a quencher dye is hybridized with a target sequence, the fluorescent dye is cleaved and separated from the complex to generate a fluorescence signal.
The detection method using the above-described fluorescent probe has an advantage of high selectivity and specificity with respect to a target sequence, but has a disadvantage that it is complex in design and expensive to use in a large amount.
The detection method using intercalating fluorescent dyes is based on DNA-binding fluorescent dyes referred to as fluorescent nucleic acid dyes or stains. Fluorescent nucleic acid dyes are advantageous because they are relatively simple molecules and therefore are easy to design and manufacture and relatively inexpensive.
However, since several criteria must be met to be used in qPCR, not all commonly known fluorescent nucleic acid dyes can be used for qPCR.
For example, fluorescent nucleic acid dyes used in qPCR should have sufficient stability during storage and PCR and should be resistant to pH range of a buffer used for PCR.
In addition, when nucleic acids are not present, the fluorescent nucleic acid dye should generate no fluorescence signal or generate only a very weak fluorescence signal, and generate a relatively strong fluorescence signal in the presence of nucleic acid.
The most commonly used dyes for the labeling of conventional nucleic acids are the following ethidium bromide:

The ethidium bromide is, for example, used (post-stained) to stain a gel that has undergone electrophoresis, and is preliminarily added in the preparation of the electrophoresis gel and used (pre-stained) for electrophoresis and staining. In addition, since ethidium bromide generates UV light of 400 nm or less and has an emission spectrum of about 620 nm when bound to DNA, it has an advantage that the presence and position of DNA can be visually confirmed easily.
In spite of the advantages of the above-mentioned ethidium bromide, ethidium bromide is a mutagen and carcinogen, which therefore requires a great deal of caution in use. In particular, research has shown that ethidium bromide interferes with the synthesis of DNA and RNA, thereby causing mutations as DNA replication is inhibited.
As a result, SYBR® green I was mainly used as a substitute fluorescent dye showing non-genotoxicity. However, since SYBR® green I inhibits the PCR process, simply increasing the concentration of SYBR® green I has the limitation that a higher maximum fluorescence signal cannot be obtained.
In other words, the intensity of the fluorescence signal increases in proportion to the concentration of SYBR® green I until a baseline concentration at which the concentration of SYBR® green I begins to significantly inhibit the PCR process. From then on, however, DNA amplification is reduced with an additional increase in concentration of the SYBR® green I. As a result, the intensity of the observed fluorescence signal is decreased, or a threshold cycle for observing the fluorescence signal of a predetermined intensity or more is increased.
It is also known that SYBR® green I is unstable under certain chemical conditions and therefore degrades considerably within a few days in a buffer solution.